Project Summary: The circadian clock controls the tinning of many important processes such as plant leaf movement and photosynthesis as well as sleep/wake cycles in humans. The molecular mechanisms controling the plant circadian clock are not well understood. Much of what is known about the core plant clock is based on genetic analysis with little attention currently given to proteins. Idenfiying protein function within the core oscillator is the next frontier of circadian research. Experiments presented in this proposal are designed to flush out the role of one such protein, LUX ARRHYTHMO, within the core clock of Arabidopsis. Isolation of a highly specific antibody for this protein will greatly assist in this endeavor. The specific aims of this proposal are to 1. Characterize LUX protein. Immunodetection experiments will be used to determine the stability and degradation of this protein. Subcellular localization of LUX will also be established. 2. Identify proteins that interact with LUX. Affinity purification and co-immunoprecipitation (Co-IP) techniques will be used to precipitate LUX and its interacting proteins. Identification of the interacting proteins will be made by mass spectrometry. 3. Determine the transcriptional targets of LUX. Chromatin immunoprecipitation (ChIP) studies will be used to identify LUX transcriptional targets by initially testing for specific candidates (i.e., CCA1, LHY, and LUX itself). A genome-wide screen for transcriptional targets will be accomplished by combining ChIP with the newly available Arabidopsis tiling array ("ChlP-chip"). Relevance: The circadian clock serves as a pace setter for a variety of biological processes, from plant leaf movement to human sleep/wake cycles. Research to elucidate the molecular mechanisms of the plant circadian clock will complement similar analyses in other systems, ultimately imparting our understanding to other circadian biologists with the potential to impact human circadian disorders.